APN is itself a target enzyme (zinc-dependent metalloprotease), not a drug that binds to a target. As a target, it is inhibited by various compounds. The following inhibitor constants (Ki) for APN are reported: Bestatin inhibits at least 12 different aminopeptidases, most with Ki below 1 μM [1]; Angiotensin IV (Ang IV) inhibits APN with pKi = 5.23 [3]; The selective APN inhibitor 7B (2(S)-benzyl-3-[hydroxy(1'(R)-aminoethyl)phosphinyl]propanoyl-L-tyrosine) has pKi = 9.22 for purified porcine APN, pKi = 8.58 ± 0.01 for recombinant human APN (from HEK293 cells) [3]. Angiotensin IV also inhibits IRAP (insulin-regulated aminopeptidase) with pKi = 6.90 for recombinant human IRAP [3].

- Enzymatic activity in striatal membranes ([3]): Striatal cell membranes were incubated with the substrate L-Leu-pNA (1.5 mM, 37°C). The Km for L-Leu-pNA was 0.238 ± 0.033 mM, Vmax was 0.007 ± 0.001 μM/min. Angiotensin IV (100 μM) inhibited up to 90% of the aminopeptidase activity (pKi = 6.09 ± 0.25). The selective APN inhibitor 7B produced a biphasic inhibition curve: a high-affinity component (pKi = 9.20 ± 0.14, accounting for 60±6% of activity, corresponding to APN) and a low-affinity component (pKi = 7.26 ± 0.10, accounting for ~40% of activity, corresponding to IRAP). [3]
- Recombinant human APN and IRAP inhibition ([3]): HEK293 cells transfected with recombinant human APN or IRAP showed inhibition by 7B with pKi values of 8.58 ± 0.01 (APN) and 7.04 ± 0.04 (IRAP). [3]

- Central blood pressure regulation ([2]): Intracerebroventricular (i.c.v.) infusion of APN inhibitors bestatin and amastatin increased arterial blood pressure and were dipsogenic in normotensive (WKY, Sprague-Dawley) and hypertensive (SHR) rat strains. I.c.v. infusion of aminopeptidase M (APN) reduced arterial blood pressure in both SHR and WKY rats. Microinfusion of APN into the paraventricular nucleus of the hypothalamus (PVN) decreased arterial blood pressure. [2]
- Renal APN and salt adaptation ([2]): In salt-resistant rat strains (Dahl SR, Sprague-Dawley), a high salt diet (8% vs. 0.8% NaCl) increased renal APN abundance and activity. In contrast, renal APN did not increase in Dahl salt-sensitive (SS) rats challenged with a high salt diet, suggesting dysregulation of APN contributes to salt sensitivity. [2]
- Striatal dopamine release ([3]): Local perfusion of Angiotensin IV (10 μM, 1 hour) into the rat striatum significantly increased extracellular dopamine levels (maximal ~150% of baseline at 60 min). Perfusion of the APN-selective inhibitor 7B alone (10, 100, or 500 nM, 1 hour) had no effect on striatal dopamine release. Co-perfusion of Ang IV (10 μM) with 7B (100 nM) potentiated the dopamine release (maximal ~350% of baseline). Co-perfusion of Ang IV (10 μM) with 7B (500 nM) completely abolished the Ang IV-induced dopamine release. [3]

- Aminopeptidase activity assay using L-Leu-pNA substrate ([3]): Membrane homogenates (50 μg protein for striatal tissue, or 2×10⁵ cells for transfected HEK293 cells) were incubated at 37°C in 96-well plates with 1.5 mM L-Leu-pNA (or indicated concentrations) and enzyme buffer (50 mM Tris-HCl pH 7.4, 140 mM NaCl, 0.1% BSA, 100 μM PMSF) alone or with test compounds. The formation of p-nitroaniline was measured by absorbance at 405 nm between 10 and 50 minutes. Rate constants were calculated by linear regression. IC50 values were calculated by non-linear regression (GraphPad Prism 4.0). pKi values were calculated using the Cheng-Prusoff equation: pKi = -log[IC50/(1+[L]/Km)], where [L] is free substrate concentration and Km is the Michaelis-Menten constant. [3]

- HEK293 cell transfection and membrane preparation ([3]): HEK293 cells were cultured in DMEM with L-glutamine (2 mM), penicillin/streptomycin, non-essential amino acids, sodium pyruvate (1 mM), and 10% fetal bovine serum at 37°C in 5% CO₂. Cells were transiently transfected with plasmid DNA (pCIneo containing human IRAP gene or pTEJ4 containing human APN cDNA) using LipofectAMINE (8 μl/ml, 1 μg/ml DNA). After 2 days, cells were harvested with 0.2% EDTA in PBS, centrifuged (500×g, 5 min, room temperature), homogenized in 50 mM Tris-HCl pH 7.4, and membranes were prepared by centrifugation (30,000×g, 30 min, 4°C). Transfected cells showed 10-fold (IRAP) and 8-fold (APN) higher enzyme activity than non-transfected cells. [3]

- Rat stereotaxic implantation and microdialysis ([3]): Male Wistar rats (250-300 g) were anesthetized with ketamine/diazepam (60/4.5 mg/kg i.p.) and placed in a stereotaxic frame. A guide cannula was implanted 3 mm above the left dorsal striatum (coordinates relative to bregma: L -2.4, A +1.2, V +2.8). A microdialysis probe (3 mm membrane length) was introduced via the cannula and perfused with modified Ringer's solution (147 mM NaCl, 4 mM KCl, 1.2 mM CaCl₂) at 2 μl/min. Animals recovered overnight. Dialysate samples (40 μl) were collected every 20 min. Drugs (Ang IV, 7B) were dissolved in modified Ringer's solution and locally administered via the probe for 1 hour. Concentrations: 7B at 10, 100, or 500 nM; Ang IV at 10 μM. Brain tissue was processed for analysis. All experiments were approved by the Ethical Committee for Animal Experiments of the Vrije Universiteit Brussel. [3]
- Intracerebroventricular infusion in rats ([2]): I.c.v. infusions of APN inhibitors (bestatin, amastatin) or aminopeptidase M were performed in normotensive (WKY, Sprague-Dawley) and hypertensive (SHR) rat strains to measure blood pressure changes. [2]
- Dietary salt manipulation in rats ([2]): Salt-resistant (Dahl SR, Sprague-Dawley) and salt-sensitive (Dahl SS) rat strains were fed high salt (8% NaCl) or low salt (0.8% NaCl) diets to assess renal APN expression and activity. [2]

- Tosedostat (CHR-2797) phase I trial in advanced solid tumors ([1]): In a phase I trial (40 patients, accelerated titration design, once daily administration), the most commonly observed toxicities were fatigue, diarrhea, peripheral edema, nausea, dizziness, and constipation. No dose-limiting toxicity was reported. One patient had a partial response (renal cell carcinoma) and four had stable disease for >6 months. [1]
- NGR-hTNF phase I trial ([1]): In a first clinical trial (16 patients, NGR-hTNF intravenously q3w at 0.2-1.6 μg/m²), the most frequent treatment-related toxicity was grade 1-2 chills (69%) occurring during first infusions. No dose-limiting toxicity occurred. [1]
- Bestatin in resected stage I squamous-cell lung carcinoma ([1]): In a randomized double-blind placebo-controlled trial (402 patients, bestatin 30 mg daily orally for 2 years), few adverse events were observed in either group. [1]

[1]. Aminopeptidase N (CD13) as a target for cancer chemotherapy. Cancer Sci. 2011 Mar;102(3):501-8.

[2]. Aminopeptidase N in arterial hypertension. Heart Fail Rev. 2008;13(3):293-298.

[3]. Involvement of insulin-regulated aminopeptidase and/or aminopeptidase N in the angiotensin IV-induced effect on dopamine release in the striatum of the rat. Brain Res. 2007;1131(1):97-105.

- Structure ([1][2]): Full-length APN consists of 967 amino acids with a short N-terminal cytoplasmic domain (8-10 amino acids), a single transmembrane helix, and a large extracellular C-terminal ectodomain containing the Zn²⁺-binding HELAH motif and the active site. APN is a non-covalently bonded homodimer with a mass of 160 kDa (2×80 kDa subunits). It is highly glycosylated in vivo. [1][2]
- Tissue distribution ([1][2]): APN is widely expressed. In the brain: highest in meninges, choroid plexus, pineal gland, paraventricular nucleus, pituitary gland, cortex, caudate-putamen, subthalamic nucleus, periaqueductal gray, thalamus, spinal cord, hippocampus, nucleus accumbens, substantia nigra, hypothalamus, raphe nucleus, pontine nucleus, inferior olive, and cerebellum granular layer. In the kidney: concentrated in renal epithelial cells (apical brush border membranes), mesangial cells, and glomeruli. [1][2]
- Role in cancer ([1]): APN is dysregulated in various solid tumors (breast, colon, lung, ovarian, pancreatic, thyroid). High APN expression is associated with tumor progression, angiogenesis, poor survival, and metastasis. Soluble APN is elevated in plasma and effusions of cancer patients compared to healthy controls. APN is a target for anti-cancer therapy (e.g., bestatin/ubenimex, tosedostat, NGR-targeted drug conjugates). [1]
- Role in hypertension ([2]): APN is involved in arterial blood pressure regulation via central (hypothalamic PVN) and renal mechanisms. A functional single nucleotide polymorphism (SNP) of the APN gene (Lys528Arg) has been associated with essential and non-modulating hypertension and reduced enzymatic activity. The APN gene maps to a quantitative trait locus (QTL) for hypertension on chromosome 1 in Dahl rats. [2]
- Role in dopamine release ([3]): In the rat striatum, APN and IRAP are present (APN accounts for ~60% of aminopeptidase activity, IRAP ~40%). Angiotensin IV increases dopamine release, which is potentiated by low-dose 7B (APN inhibition, prolonging Ang IV half-life) and abolished by high-dose 7B (both APN and IRAP inhibition). The authors hypothesize that IRAP and/or AP-N may act as receptors (not just enzymes) mediating Ang IV effects. [3]

9054-63-1

Microsomal aminopeptidase;9054-63-1

168010185

Typically exists as solid at room temperature

Produced by Aspergillus niger fermentation

1

1

10

63

1510

0

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)